Receptor-mediated endocytosis is the process by which many biologically important macromolecues including nutrients, growth factors, immunoglobulins, viruses and toxins are efficiently internalized after binding to high affinity receptors on the cell surface. Receptor-ligand complexes are clustered into specialized structures, termed clathrin- coated pits which invaginate and pinch-off to form endocytic coated vesicles carrying their membrane and soluble contents into the cell. Receptor-mediated endocytosis is driven, in part, by a cycle of assembly and disassembly of the major coat constituents, clathrin and AP complexes, which assemble from a cytoplasmic pool onto the membrane, mediate coated vesicle formation and then are recycled from coated vesicles. Our goal is to identify mechanisms which regulate each event in the clathrin-coated vesicle cycle and which mediate the sorting of receptors into coated pits. During the first period of this grant, novel cell-free assays were developed which reconstitute several biochemically distinct events in clathrin coated vesicle formation, including coat assembly, invagination, receptor sorting and coated vesicle budding. Extensive biochemical and morphological characterization of the events reconstituted in vitro have established the validity of these assays and have revealed a previously unexpected complexity in the regulation of these events. Both ATP and GTP hydrolysis are required at multiple stages in coated vesicle formation as- are cytosolic factors in addition to clathrin and AP complexes. We have identified dynamin as one of the GTPases required at an intermediate stage in coated vesicle formation. Here we propose to utilize these powerful tools for the systematic biochemical dissection of receptor-mediated endocytosis as driven by the clathrin-coated vesicle cycle. The Specific Aims for this funding period are: 1) To extend our morphological analysis of coated vesicle formation so as to establish structurally-defined correlates to the biochemically-defined intermediates detected in vivo and in vitro. 2) To exploit our rapid and quantitative stage-specific assays for the identification and purification of novel cytosolic factors required for distinct events in the clathrin-coated vesicle cycle. 3) To identify the events in receptor-mediated endocytosis which require an active dynamin GTPase by detailed phenotypic analysis of the consequences of expressing dominant-interfering dynamin mutants on coated vesicle formation and on other intracellular trafficking pathways in intact cells. 4) To identify the exact role of the dynamin GTPase through biochemical characterization of its activities in the context of receptor-mediated endocytosis and coated vesicle formation in vitro. The ambitious and intensive research program described here underscores our long-term commitment to elucidating the molecular mechanisms which govern receptor- mediated endocytosis.